Glutamine synthetase and other enzymes can be covalently modified by a variety of mixed function oxidases from bacterial and mammalian sources. It has been suggested that this oxidative modification specifically marks those enzymes for degradation. If so, then there must be proteases which recognize the modified forms and degrade them. A protease has now been detected and purified from E. coli which specifically degrades oxidized glutamine synthetase. The initial cleavage releases a 3600 MW peptide from the carboxy terminal end of the protein. Native glutamine synthetase is not a substrate. The protease purification scheme includes ammonium sulfate fractionation, DEAE cellulose chromatography at pH 7.5, hydrophobic chromatography on phenyl Sepharose and HPLC phenyl, and HPLC DEAE ion exchange chromatography at pH 9.0. The purified protease is homogeneous on SDS-polyacrylamide gel electrophoresis. The properties of this protease distinguish it from proteases previously purified from E. coli. Its molecular weight is 75,000, and it is monomeric. Optimum activity of the protease is at pH 9. Its activity is inhibited by the chelating agents EDTA and o-phenanthroline but not be diisopropylfluorophosphate or other serine protease inhibitors. ATP does not enhance its activity. In addition to degrading oxidized glutamine synthetase, the protease degrades acetylated and carboxymethylated glutamine synthetase as well as insulin and casein but not chromogenic peptide substrates of serine proteases or collagenase. The preferred cleavage sites have been determined using oxidized insulin B chain as substrate.